AM Fan, California Environmental Protection Agency, Oakland, CA, USA [607471]

Nitrate
AM Fan, California Environmental Protection Agency, Oakland, CA, USA
/C2112014 Elsevier Inc. All rights reserved.
lName: Nitrate
lChemical Abstracts Service Registry Number: 14797-55-8
lMolecular Formula: NO 3
lChemical Structure:
O
O–N+
–O
Background
Nitrate is commonly found in drinking water sources especially
in agricultural areas where nitrogen fertilizer is used, and where
unregulated shallow private wells are more at the risk of
contamination. The World Health Organization (WHO)guideline of 50 ppm and the US maximum contaminant level
(MCL) of 45 ppm for nitrate in drinking water have been
established for protecting infants from methemoglobinemia,commonly known as blue baby syndrome. The health protec-tive value continues to be a subject of public health interest for
many years, with varying opinion on whether it is too high or
too low. Evaluation of nitrate will need to include consider-ation of nitrite because both are closely related in the nitrogen
cycle in the environment and the body, and nitrite plays
a major role in inducing toxicity after its formation fromnitrate. More recently, reports of nitrate in drinking water,
especially at levels higher than 50 ppm, have been associated
with other health effects other than methemoglobinemia. Thistoxicological review provides an update on the health effects of
nitrate with a focus on methemoglobinemia, reproductive and
developmental effects, potential carcinogenicity, and especiallyendocrine/thyroid effects.
Uses
Nitrate is used in fertilizers; in the manufacture of nitrites,nitrous oxide, explosives, pyrotechnics, matches, freezingmixtures, and special cements; as a coloring agent and
preserving additive in food; for coagulation of latexes; in the
nuclear industry; and for odor (sulfi de) and corrosion control
in aqueous systems.
Environmental Behavior, Fate, Routes, and Pathways
Nitrate (NO 3/C0), a product of nitrogen oxidation, is a naturally
occurring ion in the environment and integrated into complexorganic molecules such as proteins and enzymes required by
living systems. Nitrate is a more stable form of oxidized
nitrogen than nitrite; however, it can be reduced by microbialaction to nitrite, which, in turn, can be reduced to variouscompounds or oxidized to nitrate by chemical and biologicalprocesses. Nitrates occur naturally in soil from microbial
oxidation of ammonia derived from organic nitrogenous
materials such as plant proteins, animals, and animal excreta.Other source contributions are wastewater, septic tank runoffs,
airborne nitrogen compounds emitted by industry and auto-
mobiles, nitrogen fertilizer, and manure from animal feeding.Nitrate in groundwater is generally found below 10 ppm, with
higher levels in areas of high agricultural activities.
Exposure and Exposure Monitoring
The contribution of drinking water to nitrate intake is usually
less than 14%, food and drinking water are the major sources
of exposure, especially food (green vegetables and cured meat)
unless the water level is higher than 45 ppm. Overall, for anaverage adult consumer who lives in an area with high water
contamination, total exposure to nitrate from food and
water is estimated to be about 60 –90 mg per person per day,
of which at least 90% is from food. The intake of nitrate may
reach 200 mg per person per day for a high vegetable consumer
or when the water is higher than the MCL. For infants, an averagedaily intake of nitrates from consumption of vegetable-basedfoods was reported to be 7.8 mg. For bottle-fed infants, intake
from milk formula made with water containing 50 mg l
/C01
nitrate would average about 8.3 –8.5 mg nitrate kg/C01day/C01.
Toxicokinetics
Nitrate is widely distributed in the body via the nitrogen cycle.
Nitrate ingestion is followed by endogenous nitrate synthesis,release from blood to saliva, conversion of nitrate to nitrite bybacteria in saliva, conversion of nitrite to nitrate in blood, and
ultimate excretion mainly as nitrate in the urine. Bioavailability
from food or drinking water is at least 92%. Absorbed nitrate israpidly transported via the blood. Plasma nitrate is dose-
dependently secreted by the salivary gland via an active trans-
port mechanism shared with iodide and thiocyanate. Thisamounts to approximately 25% of the ingested nitrate in saliva,
or 10 times the concentration in plasma. Nitrate is secreted by
passive diffusion into breast milk.
Of toxicological concern is the metabolism of nitrate to
nitrite and N-nitroso compounds. Approximately 5 –20% of
the ingested nitrate is reduced by oral bacteria to nitrite, and thein vivo nitrite formed represents 80% of total nitrite exposure.
Nitrate biosynthesis appears to be from nitric oxide to N
2O3
and the reaction of N 2O3with water to yield nitrite. Nitrite is
rapidly oxidized to nitrate through reaction with hemoglobin.Other cell types can also form nitric oxide, generally from
arginine. Ingested nitrate can result in the formation of N-
nitroso compounds with concomitant ingestion of nitrosation
cofactors and precursors (e.g., protein). Such endogenous
Encyclopedia of Toxicology, Volume 3 http://dx.doi.org/10.1016/B978-0-12-386454-3.01067-8 523

nitrosation can occur in the stomach, small intestine, and other
parts of the body where the bacterial flora reduce nitrate to
nitrite. Active secretion of nitrate and conversion of nitrate to
nitrite in saliva occur in humans and most laboratory species
except rats.
The endogenous production of nitrate and its role in the
nitric oxide pathway is bene ficial for protecting against oral and
gastrointestinal diseases and also for its role in vascular fitness
and exerting antihypertensive effects.
Mechanism of Toxicity
The acute oral LD 50values for sodium nitrate range from 2480
to 9000 mg kg/C01in rats, mice, and rabbits. Acute, subchronic,
and chronic animal toxicity studies showed low toxicity for
nitrate as sodium or potassium nitrate. A long-term study
showed a slight depression in growth rate. Nitrite, but notnitrate, is capable of inducing methemoglobinemia ( see
Nitrites, for more details).
Nitrate has been reported to be associated with thyroid
effects in experimental animals and humans. Possible mode of
action includes inhibition of iodine uptake to thyroid, serum
T
3and T 4changes, and tissue T 3changes. However, there is
a lack of knowledge on the differences in the mode of action to
permit animal-to-human extrapolation. While the data indi-
cate humans and rats exhibit similar dose –response relation-
ships in acute inhibition of thyroidal iodide uptake, they showdifferences in thyroid hormone response following iodide
uptake inhibition. Comparative data are needed for serum and
brain tissue levels of thyroid hormones and characterization ofthe dose –response relationship between changes of thyroid
hormone levels and adverse effects.
Early experimental and field studies in mammals have
found inorganic nitrate to be goitrogenic. The effects were
observed in rats following oral and parenteral administration
of potassium and sodium nitrate, whereas antithyroid effectswere also reported in sheep and pigs administered potassiumnitrate. Nitrate exposure through diet or drinking water caused
functional and histological changes to the thyroid gland in rats
and pigs. More recent investigations between 2000 and 2010reported changes in thyroid and thyroid activity following
exposure to nitrate. In these more recent studies, nitrate expo-
sure has consistently resulted in increases in thyroid weightand/or changes to the follicle cell; however, the reported
thyroidal hormone changes have not been as consistent. The
studies reported increased thyroid weights with a decrease inthyroid hormones (i.e., T
3and T 4) and/or decrease in thyroid
stimulating hormone. However, not all the results are consis-tent with the expected outcome of a sodium –iodide symporter
(NIS) inhibitor, which can be seen as supplementation ofiodine in the diet that did not result in thyroid changes.
Overall, the data support that nitrate impairs thyroid function
involving the hypothalamic –pituitary –adrenal axis.
Acute and Short-Term Toxicity
The major acute toxicity concern is methemoglobinemiaafter oral ingestion. Ingested nitrate is reduced to nitrite in thegastrointestinal tract and binds to hemoglobin to formmethemoglobin. Nitrite absorbed in the blood stream isinvolved in the oxidation of hemoglobin to methemoglobin
in which the iron has been oxidized from the ferrous to the
ferric state.
NO
2/C0țoxyhemoglobin/C0
Fe2ț/C1
/methemoglobin/C0
Fe3ț/C1
țNO/C0
3
Nitrate is a competitive inhibitor of iodide uptake to
thyroid at the NIS. It binds to NIS on the surface of thyroid
follicular cells that can result in depression of thyroid
hormone depression. It is believed to have a common modeof action with some other contaminants. In rats tested for
radioactive iodine uptake, nitrate was found to be the least
potent when compared with perchlorate and thiocyanate ona molar basis. Perchlorate was 10 times more potent thanthiocyanate and 300 times more potent than nitrate. When
the relative potencies of the three anions were determined in
Chinese hamster ovary cells, perchlorate inhibited
125I/C0
uptake at the NIS at 15, 30, and 240 times that of thiocyanate,
iodide, and nitrate, respectively. It was noted that the results
are consistent with a common mode of action by these anionsof simple competitive interaction, in which any one of the
anions, either individually or in a mixture of the three, is
indistinguishable from a concentration or dilution of eitherone of the remaining two ions in inhibiting iodine uptake at
the NIS.
The available data indicate that humans and rats exhibit
similar dose –response relationships in terms of acute inhibi-
tion of thyroidal iodide uptake, but there are notable differ-ences in terms of thyroid hormone response, which lead to
iodide uptake inhibition. When dose –response data for
changes in serum T
3,T4, and thyroid stimulating hormone
(TSH) levels from studies in humans, rats, mice, and rabbitswere analyzed, thyroid homeostasis in the rat appears to bemuch more sensitive to perchlorate than the other species. Rats
showed an increase in serum TSH at 0.1 mg kg
/C01day/C01,
whereas other species were still unresponsive at
10 mg kg/C01day/C01. Less pronounced but consistent effects were
seen with serum T 3and T 4. These cross-species comparisons
provide a basis for special consideration in evaluating ratstudies for their relevance to humans.
Chronic Toxicity
Associations of nitrate exposure and thyroid effects in humanshave been reported in the literature since 1994, and are thesubject of ongoing investigations and research. The consump-
tion of drinking water containing nitrate at levels higher than
50 ppm has been associated with: (1) increased thyroid volumeand subclinical thyroid disorders (thyroid hypoechogenicity by
ultrasound, increased serum thyrotropin level, and positive
thyroperoxidase antibodies in school children in Eastern Slo-vakia); (2) increased thyroid volume in healthy women in the
Netherlands; (3) increased incidence of goiter in children in
villages of Bulgaria; and (4) increased relative risk of thyroiddisorder and goiter rates in pregnant women in Bulgaria.
In a cohort study of 21 977 women in Iowa, the authors524 Nitrate

reported an increased risk of thyroid cancer with higher average
nitrate levels in water and with longer consumption of waterexceeding 22 ppm nitrate, but no association between nitrate in
drinking water and prevalence of hypothyroidism or hyper-
thyroidism. Increased dietary nitrate was also associated withan increased risk of thyroid cancer and with the prevalence of
hypothyroidism.
Studies that did not report a relationship between nitrate
and thyroid changes included: (1) a study in 10 adultvolunteers given sodium nitrate in drinking water that found
no effect on thyroidal I
131uptake and plasma concentrations
of thyroid hormones (T 3,r T 3,T4, and TSH); (2) a study in
3059 clinically healthy people aged 18 –70 from different
regions of Germany that found no in fluence of urinary
nitrate excretion on the prevalence of goiter; (3) a study of3772 subjects aged 20 –79 from Pomerania/Germany that
showed no diagnosed thyroid disorder; (4) a study of infantsthat found no association between cord blood levels ofnitrate, thiocyanate, and perchlorate anions and newborn
weight, length, and head circumferences; and (5) an analysis
of the urinary levels of nitrate, perchlorate, and thiocyanate;urinary iodide concentration; serum levels of thyroid
hormones (T
4, TSH), albumin, cotinine, and c-reactive
protein in 2299 men and women (aged 12 years and higher,
noninstitutionalized) in the 2001 –02 US National Health
and Nutrition Examination Survey.
Reproductive Toxicity
In four teratogenicity studies by the US Food and DrugAdministration (FDA), sodium and potassium nitrate were
given orally at four dose levels to pregnant hamsters, mice, rats,
and rabbits. No teratogenicity, soft or skeletal tissue abnor-malities, or other effects were observed on nidation, maternal
or fetal survival, fetal toxicity, malformations, or maternal
reproductive effects. Testicular toxicity has been reported in ratsand mice following subchronic exposure to nitrate.
Epidemiological studies have suggested an association
between exposure to nitrate in drinking water and spontaneousabortions, intrauterine growth restrictions, and birth defects,but no clear exposure –response relationship can be estab-
lished. A case –control study in New Brunswick, Canada
examined the relationship between maternal exposure tonitrates in drinking water and risk of delivering an infant with
a central nervous system malformation. A population-based
case –control study in California investigated the association
between maternal periconceptional exposure to nitrate from
drinking water and diet and risk for neural tube defects. A study
investigated the relationship between community drinkingwater quality and spontaneous abortion in patients whoentered Boston Hospital for Women Division of Brigham and
Women’ s Hospital in Massachusetts. A case –control study in
South Australia investigated malformation (mainly neural tube
defects and defects affecting multiple systems) in women and
association with drinking water consumption from speci fic
sources differing in nitrate content. A case –control study in the
Mount Gambier region of South Australia investigated therelationship between mothers ’maternal drinking water source
and malformations in offspring as a follow-up study. A case –control study in Prince Edward Island, Canada investigated
the dose –response relationship between nitrate level and
intrauterine growth restriction and prematurity. A case –control
study examined all deliveries in Sweden for neural tube defects.A retrospective cohort study in Ostergotland County, Swedeninvestigated the association between water nitrate at or greater
than 2 mg l
/C01and cardiac malformation using a geographic
information system to link periconceptional or early pregnancy
address to water supplies. A case –control study of counties
along the Texas –Mexico border tested the association between
increased water nitrate and spina bi fida and anencephaly.
A cross-sectional study in South Africa investigated the associ-
ation between water from high nitrate regions and prematurity
or size of South West Africa/Namibian infants based onsamples taken from wells used at the time of home visit.A study investigated clusters of spontaneous abortion reported
in Le Grange County, Indiana, after which the water was tested
for nitrate.
Genotoxicity
Genotoxicity studies showed mostly negative responses fornitrate.
Carcinogenicity
Sodium and potassium nitrate have been tested for potential
carcinogenicity, alone and in combination with nitrosatable
compounds. Nitrosating agents can be ingested from foodand drinking water, and synthesized from ingested nitrate
and nitrite. Nitrosating agents can react under certain
conditions with nitrosatable compounds to form N-nitrosa-
mines and N-nitrosamides, some of which are animal
carcinogens. Nitrosating agents (e.g., nitrous acid and nitrousanhydride) that arise from nitrite under acidic gastricconditions can react with amines or amides to form nitro-samines or nitrosamides, and the induction of tumors in
animals via endogenous synthesis of N-nitroso compounds
has been demonstrated. Nitrosamines need to be activated
metabolically by cytochrome P450 enzymes to electrophilic
intermediates to exert a carcinogenic effect, while nitro-
samides are direct-acting carcinogens. Ascorbic acid is aninhibitor of nitrosation reactions. It has been shown to lower
the incidence of tumors in animal experiments, and reduce
the risk for cancer that is associated with ingested nitrite inepidemiological studies.
NTP chronic bioassay studies in rats and male mice did not
show carcinogenicity where nitrate was administered alone indrinking water or diet (three studies in mice and four studies inrats) or was coadministered with nitrosatable compounds, and
when nitrite was given alone in the diet by gavage or in the
drinking water to rats and mice.
Various ecological studies, case –control studies, and cohort
studies conducted worldwide on the relationship betweenhuman exposure to nitrate and the risk for various cancers re-ported inconsistent results. These were reviewed by the Inter-
national Agency for Research in Cancer (IARC) and included
studies of: (1) cancers of the colon, liver, pancreas, and rectumNitrate 525

in Canada, China, Slovakia, and Thailand; (2) leukemia and
lymphoma in Canada, China, Egypt, Finland, Italy, Slovakia,the United Kingdom, and the USA; (3) gastric and esophageal
tumors in China, Columbia, Costa Rica, Denmark, Japan,
Netherlands, Poland, Scotland, Spain, Sweden, the UnitedKingdom, and the USA; (4) tumors of the nervous system,
mainly brain, in Australia, Canada, England, France, Germany,
Israel, and the USA; and (5) genital and urinary tract tumors inDenmark, Egypt, Germany, Slovakia, Spain, and the USA. An
increased risk of non-Hodgkin ’s lymphoma and urinary
bladder was reported in some studies but not others at similar
exposure levels of nitrate in drinking water. Meta-analysis of
prospective, case –control, and cohort studies reported greater
risks for colorectal cancer associated with consumption of
processed meat.
IARC concluded that ingested nitrate under conditions
that result in endogenous nitrosation is probably carcinogenic
to humans (Group 2A). The underlying mechanism for the
carcinogenicity determination is endogenous nitrosation that
results in the formation of N-nitroso compounds, some of
which are known carcinogens. There is an active endogenous
nitrogen cycle in humans wherein nitrosating agents that
arise from nitrite under acidic gastric conditions react readily
with nitrosatable compounds, especially secondary aminesand amides, to generate N-nitroso compounds. These nitro-
sating conditions are enhanced following ingestion of addi-tional nitrate, nitrite, or nitrosatable compounds. Speci fically,
for nitrate alone, IARC found that there is inadequate evidence
in humans for the carcinogenicity of nitrate in food. There is
inadequate evidence in humans for the carcinogenicity of
nitrate in drinking water. There is inadequate evidence in
experimental animals for the carcinogenicity of nitrate. Fornitrite, in food, IARC found that it is associated with an
increased incidence of stomach cancer. There is sufficient
evidence in experimental animals for the carcinogenicity of
nitrite in combination with amines or amides. There is limited
evidence in experimental animals for the carcinogenicity of
nitrite per se.
More recent studies after the IARC evaluation did not
report an association between nitrate in water and non-Hodgkin lymphoma, breast, bladder, colon, urinary, andpancreatic cancer. Studies on dietary intake of nitrate/nitrite
reported some associations with increased risk of bladder
cancer, esophageal squamous cell carcinomas, colorectalcancer, and thyroid cancer. Studies in Iowa reported no
association to renal cell carcinoma ( >5 and >10 ppm nitrate-
N); no association with non-Hodgkin lymphoma (below
3 ppm); and increased risk of thyroid cancer in older women
(>5 ppm nitrate for 5 years or longer, relative risk ¼2.6, 95%
confidence interval ¼1.6–6.2) (no association with preva-
lence of hypothyroidism or hyperthyroidism). Study on die-
tary intake of nitrate and nitrite (National Institutes of
Health –American Association of Retired Persons Diet and
Health Study) suggested a role and further studies on ovarian
and thyroid cancer risk and pancreatic cancer in men.
Overall, interpretation of the data is complicated by various
factors such as the amount of nitrate/nitrite ingested, theconcomitant ingestion of nitrosation cofactors and precursors,
specifi c factors that increase nitrosation, and some study
limitations.Carcinogenicity
Carcinogenicity is a possible endpoint of concern because
of the biological plausibility of endogenous nitrosation of
ingested nitrate and nitrite, with the conversion of nitrate to
nitrite, and formation of genotoxic/carcinogenic N-nitroso
compounds, such as N-nitrosamines and N-nitrosamides,
some of which are known carcinogens. The mechanism mayinvolve acid-catalyzed (e.g., in acidic stomach) or cell-mediated(such as bacteria and immune cell, neutral pH) formation. Inhumans, nitrite swallowed in saliva is usually first converted in
the stomach to nitrous acid (HNO
2) which is spontaneously
converted to the active nitrosating species nitrous anhydride(N
2O3). Nitrous anhydride is a strong nitrosating agent, which
donates NOțto secondary and tertiary amines to form nitro-
samines. Nitrous acid can also be protonated to nitrous acid-ium ion, which reacts directly with neutral amides to form
nitrosamides. About 40 –70% of total human exposure to N-
nitroso compounds is from endogenous formation.
Clinical Management of Methemoglobinemia
People may have lifelong methemoglobinemia and can beasymptomatic at relatively high levels. Methemoglobinemia is
a side effect of nitric oxide therapy for acute respiratory stresssyndrome and persistent hypertension in newborns. Rapidity
of methemoglobin formation may lead to severe symptoms.
Acquired methemoglobinemia can also result from exposure toother chemicals and pharmaceuticals. People with glucose-6-
phoshate dehydrogenase de ficiency and infants aged 6 months
and less are at increased risk. Methemoglobinemia should be
suspected in patients with central cyanosis and low oxygensaturations, which are unresponsive to oxygen therapy. Treat-
ment should be guided by the severity of the symptoms
initially, aimed at decreasing the level of methemoglobinfound in the blood, and accompanied by removal from expo-
sure. Methylene blue is used when signi ficant symptoms are
present (dizziness, confusion, seizure, somnolence, and head-
ache), with clinical observation of lowering methemoglobin
levels. However, methylene blue will not be responsive in
patients with G6PD de ficiency, and it has not been FDA
approved for the pediatric population.
Other Health Effects
Associations between nitrate in drinking water and hyperten-sion, immunologic effects, recurrent respiratory tract infection,diarrhea, and childhood onset of diabetes mellitus have been
reported. The data are very limited and not convincing, and
results were con flicting in studies of childhood diabetes mellitus.
Exposure Standards and Guidelines
The WHO guideline for nitrate in drinking water is 50 ppm.
The US MCL for nitrate in drinking water is 45 ppm as nitrate,
or 10 ppm as nitrate-N, and 1 ppm for nitrite as nitrite-N.The combined MCL is 10 ppm nitrate/nitrite-N.526 Nitrate

See also: Ammonium Nitrate; Butyl Nitrite; Nitrites; Amyl Nitrite;
Nitrite Inhalants.
Further Reading
Aly, H.A., Mansour, A.M., Abo-Salem, O.M., Abd-Ellah, H.F., Abdel-Naim, A.B., 2010.
Potential testicular toxicity of sodium nitrate in adult rats. Food Chem. Toxicol. 48
(2), 572 –578.
Aschebrook-Kilfoy, B., Cross, A., Stolzenberg-Solomon, R.Z., Schatzkin, A.,
Hollenbeck, A.R., Sinha, R., Ward, M.H., August 1, 2011. Pancreatic cancer and
exposure to dietary nitrate and nitrite in the NIH-AARP diet and health study. Am. J.
Epidemiol. 174 (3), 305 –315.
Aschebrook-Kilfoy, B., Ward, M.H., Gierach, G.L., Schatzkin, A., Hollenbeck, A.R.,
Sinha, R., Cross, A.J., 2012. Epithelial ovarian cancer and exposure to dietarynitrate and nitrite in the NIH-AARP Diet and Health Study. Eur. J. Cancer Prev. 21(1), 65 –72.
Eskiocak, S., Dundar, C., Basoglu, T., Altaner, S., 2005. The effects of taking chronic
nitrate by drinking water on thyroid functions and morphology. Clin. Exp. Med. 5,66–71.
Fan, A.M., 2011. Nitrate and nitrite in drinking water: a toxicological review. In: Nria-
gu, J.O. (Ed.), Encyclopedia of Environmental Health, vol. 4. Elsevier, Burlington,pp. 137 –145.Fan, A.M., Steinberg, V.E., 1996. Health implications of nitrate and nitrite in drinking
water: an update on methemoglobinemia occurrence and reproductive anddevelopmental toxicity. Reg. Toxicol. Pharmacol. 23, 35 –43.
Kilfoy, B.A., Zhang, Y., Park, Y., Holford, T.R., Schatzkin, A., Hollenbeck, A.,
Ward, M.H., 2011. Dietary nitrate and nitrite and the risk of thyroid cancer in the
NIH-AARP Diet and Health Study. Int. J. Cancer 129 (1), 160 –172.
L’hirondel, J., L ’hirondel, J.L., 2001. Nitrate and Man, Toxic, Harmless or Bene ficial?
CABI Publishing, Wallingford, UK .
Ward, M.H., Kilfoy, B.A., Weyer, P.J., Anderson, K.E., Folsom, A.R., Cerhan, J.R.,
2010. Nitrate intake and the risk of thyroid cancer and thyroid disease. Epide-
miology 21 (3), 389 –395.
Zaki, A., Ait Chaoui, A., Talibi, A., Derouiche, A.F., Aboussaouira, T., Zarrouck, K.,
Chait, A., Himmia, T., 2004. Impact of nitrate intake in drinking water on thethyroid gland activity in male rat. Toxicol. Lett. 147, 27 –33.
Relevant Website
http://www.who.int/water_sanitation_health/dwq/chemicals/nitratenitrite2ndadd.pdf –
Background Document for Development of WHO Guidelines for Drinking-waterQuality. World Health Organization, Geneva, Switzerland.Nitrate 527